AgRP neurons exert remarkable control over hunger. They are activated when stores are reduced, and once engaged, they induce intense hunger. Of great interest are the means by which AgRP neuron activity is controlled. While circulating hormones like leptin and ghrelin have direct effects on AgRP neurons, AgRP neurons also receive extensive neural input. This latter point has three important implications. First, changes in AgRP neuron activity in response to fasting could primarily be caused by alterations in the strength and number of afferent synapses (i.e. synaptic plasticity). Indeed, an important role for synaptic plasticity has already been established. Second, in addition to effects on plasticity, the fasted state is also likely sensed directly or indirectly by the afferent neurons themselves, with this information then being transmitted to AgRP neurons through the very same synapses. Third, cues other than those related to energy balance could also engage these afferents to bring about rapid changes in AgRP neuron activity. Of note, food-related cues, without any consumption of food, have recently been shown by others, and us, to rapidly reduce AgRP neuron activity. The existence of such rapid, non-homeostatic control of AgRP neurons has important implications, and is highly likely to be mediated by afferent neural input. This goal of this grant is to study mechanisms by which AgRP neuron activity is controlled. Aim 1 will focus on synaptic plasticity and determine how fasting upregulates dendritic spines and excitatory synaptic activity - an important means of control discovered during the previous cycle. In preliminary studies, we demonstrate that an AMPK ? p21-activated kinase (PAK) pathway is key. We propose the following mechanism: increased Ca2+ in AgRP neurons (due to NMDAR activation, increased AgRP neuron firing and likely also ghrelin) ? CaMMK? ? AMPK ? p21-activated kinase (PAK) ? excitatory plasticity. To test this we are using 2P imaging and a genetically encoded FRET-based sensor to image AgRP neuron AMPK activity moment-to-moment, both in brain slices and in awake behaving mice, in response to various perturbations. Aim 2 will use single neuron RNA-seq to a) create a transcriptional atlas of all neurons residing in the arcuate nucleus (using Drop-seq), b) assess the transcriptional signature of AgRP neurons in comparison to other ARC neurons, and also probe for transcriptional heterogeneity between subsets of AgRP neurons, c) assess the transcriptional effects of fasting and leptin on individual AgRP neuron gene expression using an innovative single neuron nuclei RNA-seq strategy designed to preserve in vivo states of gene expression, and d) develop a single neuron nuclei RNA- seq technique to transcriptionally identify rabies+ AgRP neuron afferents. Finally, Aim 3 will employ an innovative 2-synapse rabies strategy to identify the anatomic sites/neurons that engage the orexigenic PVHglutamatergic neuron ? AgRP neuron circuit discovered during the previous cycle. Our ultimate goal in this Aim is to identify the information carried by these afferents.